Modulation of ghrelin levels and ghrelin/unacylated ghrelin ratio using unacylated ghrelin

ABSTRACT

A method and a composition for decreasing ghrelin levels and/or decreasing ghrelin/unacylated ghrelin ratio in a subject, the method comprising administering an effective amount of unacylated ghrelin, a fragment thereof, an analog thereof and/or pharmaceutically acceptable salts thereof to the subject wherein a reduction in ghrelin levels and/or a reduction in ghrelin/unacylated ghrelin ratio is beneficial to the subject. Also, use of ghrelin level and/or ghrelin/unacylated ghrelin ratio as biomarkers for determining a subject&#39;s likelihood of responding to and/or benefiting from administration of unacylated ghrelin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. provisionalpatent application No.

61/576,217, filed Dec. 15, 2011, the content of which is hereinincorporated in its entirety by reference.

FIELD OF THE INVENTION

This invention relates to the use of unacylated ghrelin, fragmentsand/or analogs thereof for modulating ghrelin levels and/orghrelin/unacylated ghrelin ratio in a subject wherein such modulation isbeneficial to the subject. The invention also relates to the compositioncomprising unacylated ghrelin, fragments and/or analogs thereofmodulating ghrelin levels and/or ghrelin/unacylated ghrelin ratio in asubject wherein such modulation is beneficial to the subject. Theinvention further relates to the use of ghrelin level and/orghrelin/unacylated ghrelin ratio as biomarkers for determining asubject's likelihood of responding to and/or benefiting fromadministration of unacylated ghrelin.

BACKGROUND

Ghrelin (also referred as “acylated ghrelin” or abbreviated as “AG”) isa 28 amino acid peptide, purified and identified from rat stomach andcharacterized by the presence of an n-octanoyl modification on the Ser3residue (Ref. 1). Acylation of ghrelin is catalyzed by the enzymeghrelin O-acyl transferase (GOAT). The expression of GOAT is mostly inthe stomach and intestine. Ghrelin is the endogenous ligand of thegrowth hormone (GH) secretagogue receptor (GHSR-1a) (Refs. 2, 3).Ghrelin is now mostly recognized as a potent orexigenic factorstimulating food intake and modulating energy expenditure (Refs. 4, 5and 6). At the peripheral level, Ghrelin exerts probably its majorphysiological action regulating glucose and lipid metabolism (Ref. 7).In fact, ghrelin has a diabetogenic action (Ref. 8) and suppressesglucose-stimulated insulin secretion and deteriorates glucose tolerance(Ref. 9). As such, elevated plasma ghrelin is of relevance in certaindisorders of the metabolism and growth such as in diabetes and obesity.Elevated plasma ghrelin levels have also been demonstrated amongstadults and children with Prader-Willi Syndrome (PWS) (Ref. 10 and 11).PWS is a genetic obesity syndrome associated in most patients with GHdeficiency. Children with PWS present a rapid weight gain along with avoracious appetite. Studies on the involvement of ghrelin in PWS haveprovided a significant rationale that the hyperphagia observed in PWS ispositively correlated with elevated ghrelin levels, consistent with theknown orexigenic effect of ghrelin (Ref. 12).

Unacylated ghrelin (also referred as “des-acyl ghrelin” or abbreviatedas “UAG”), is the non-acylated form of ghrelin. Its concentration inplasma and tissue is higher compared to ghrelin. UAG has long beenconsidered as a product with no physiological role as it fails to bindthe only known ghrelin receptor GHSR-1a at physiological concentrationsand has no physiological effect on GH secretion (Ref. 15). However, UAGis a biologically active peptide, particularly at the metabolic leveland its administration has been shown to induce a negative energybalance by decreasing food intake and delaying gastric emptying (Ref.16). Over-expression of UAG in mice results in a decrease in fataccumulation with an increase in insulin sensitivity and glucosetolerance (Refs. 16 and 17).

UAG has been shown to prevent the hyperglycemic effects of ghrelin, whenadministered concomitantly, in healthy volunteers, see in particularU.S. Pat. No. 7,825,090, herein incorporated in its entirety byreference. This initial observation was followed by several reports onthe anti-diabetogenic potential of UAG (Refs. 18, 19, 30, 31 and 32).

In vitro, in vivo and clinical evidence indicate that UAG prevents thediabetogenic effects of ghrelin in healthy volunteers and inGH-deficient patients (Refs. 18 and 19). It inhibits both basal andghrelin-induced glucose secretion by human hepatocytes (Ref. 31). Inrats, UAG enhances portal insulin response to glucose (Ref. 32) andreduces fat deposition and triglycerides levels, as observed intransgenic mice overexpressing UAG (Ref. 16). In vitro, UAG stimulatesinsulin secretion from insulinoma cells (Ref. 32) and promotesproliferation and inhibits apoptosis of beta cells (Ref. 33).

The anti-diabetogenic effects and ghrelin-antagonizing effects of UAG,fragments and analogs thereof have been reported in U.S. Pat. No.7,485,620; U.S. Pat. No. 8,222,217; U.S. Pat. No. 8,318,664 and in WO2008/145749, which are all in their entirety incorporated herein byreference.

Recent experiments on circulating angiogenic cells (CAC) indicates thatUAG beneficially impacts the vascular remodeling process which is knownto be impaired in type 2 diabetes patients. The effects of UAG on CAChave been reported in U.S. Patent Application Serial Number 2010/0016226and in WO 2009/150214, herein incorporated in their entirety byreference.

Obese mice and humans have been reported to present lower UAG levelsthan normal weight subjects, indicating that obesity might be correlatedwith a relative UAG deficiency (Refs. 34, 35 and 21). It has beenobserved that insulin-resistant obese subjects have an elevated AG/UAGratio when compared to insulin-sensitive obese subjects (Refs. 20 and22).

Treatments that target ghrelin and the GHS-R (i.e., ghrelin antagonists)have been suggested as attractive pharmacologic avenues to fight againstobesity and other conditions, disorders and diseases associated withghrelin. Several GHS-R ligands and anti-obesity vaccines have beenproposed (Ref. 24). Other pharmacological approaches inducing antibodiesagainst ghrelin, ghrelin enantiomers and inhibition of ghrelinacyl-transferase (GOAT) (Ref. 25) have been investigated; however, dueto lack of efficacy, non-selectivity and lack of sustained weight loss,these pharmacological approaches have not yet reached the market (Ref.26).

Therefore, there exists a need in the art for an efficient and moredirect way of modulating circulating ghrelin levels and/or circulatingghrelin/unacylated ghrelin ratio in subjects wherein such modulation isbeneficial to the subject and for more efficient ways of identifyingthose subjects that can benefit from modulation of ghrelin levels andghrelin/unacylated ghrelin ratio.

SUMMARY OF THE INVENTION

According to one aspect, the present invention provides a method fordecreasing ghrelin levels in a subject, comprising administering aneffective amount of unacylated ghrelin, a fragment thereof, an analogthereof and/or pharmaceutically acceptable salts thereof to the subject.

According to another aspect, the present invention provides a method fordecreasing ghrelin levels and ghrelin/unacylated ghrelin ratio in asubject, comprising administering an effective amount of unacylatedghrelin, a fragment thereof, an analog thereof and/or pharmaceuticallyacceptable salts thereof to the subject.

According to another aspect, the present invention provides a method forameliorating a symptom associated with ghrelin levels in a subject,comprising administering an effective amount of unacylated ghrelin, afragment thereof, an analog thereof and/or pharmaceutically acceptablesalts thereof to the subject.

According to another aspect, the present invention provides a method forameliorating a symptom associated with ghrelin levels and withghrelin/unacylated ghrelin ratio in a subject, comprising administeringan effective amount of unacylated ghrelin, a fragment thereof, an analogthereof and/or pharmaceutically acceptable salts thereof to the subject.

According to another aspect, the present invention provides a method forameliorating and/or treating a condition caused by elevated ghrelinlevels in a subject, comprising administering to a subject having thecondition an effective amount of unacylated ghrelin, a fragment thereof,an analog thereof and/or pharmaceutically acceptable salts thereof.

According to another aspect, the present invention provides a method forameliorating and/or treating a condition cause by elevated ghrelinlevels and elevated ghrelin/unacylated ghrelin ratio in a subject,comprising administering to a subject having the condition an effectiveamount of unacylated ghrelin, a fragment thereof, an analog thereofand/or pharmaceutically acceptable salts thereof.

According to another aspect, the present invention provides a method forpreventing weight gain in a subject following diet-induced weight loss,comprising administering an effective amount of unacylated ghrelin, afragment thereof, an analog thereof and/or pharmaceutically acceptablesalts thereof to the subject.

According to another aspect, the present invention provides apharmaceutical composition comprising an effective amount of unacylatedghrelin, a fragment thereof, an analog thereof and/or pharmaceuticallyacceptable salts thereof and a pharmaceutically acceptable diluent fordecreasing ghrelin levels in a subject.

According to another aspect, the present invention provides apharmaceutical composition comprising an effective amount of unacylatedghrelin, a fragment thereof, an analog thereof and/or pharmaceuticallyacceptable salts thereof and a pharmaceutically acceptable diluent forameliorating symptoms associated with ghrelin levels in a subject.

According to another aspect, the present invention provides apharmaceutical composition comprising an effective amount of unacylatedghrelin, a fragment thereof, an analog thereof and/or pharmaceuticallyacceptable salts thereof and a pharmaceutically acceptable diluent forpreventing weight gain in a subject following diet-induced weight loss.

According to another aspect, the present invention provides the use ofan effective amount of unacylated ghrelin, a fragment thereof, an analogthereof and/or pharmaceutically acceptable salts thereof and apharmaceutically acceptable diluent for decreasing ghrelin levels in asubject.

According to another aspect, the present invention provides the use ofan effective amount of unacylated ghrelin, a fragment thereof, an analogthereof and/or pharmaceutically acceptable salts thereof and apharmaceutically acceptable diluent for ameliorating symptoms associatedwith ghrelin levels in a subject.

According to another aspect, the present invention provides the use ofan effective amount of unacylated ghrelin, a fragment thereof, an analogthereof and/or pharmaceutically acceptable salts thereof and apharmaceutically acceptable diluent for preventing weight gain in asubject following diet-induced weight loss.

According to another aspect, the present invention provides a method forscreening within a population of subjects suffering from diabetes,Prader-Willi Syndrome, obesity, insulin resistance or hyperphagia, whichsubjects within the population are susceptible of benefiting from anadministration of unacylated ghrelin, a fragment thereof or an analogthereof, comprising: determining a level of circulating ghrelin from thesubjects of the population; processing the level of circulating ghrelinat least in part based on a reference level of circulating ghrelin toderive information conveying whether the level of circulating ghrelin iselevated; and causing conveyance of the information to a recipient fordetermining the subject's susceptibility of benefiting fromadministration of unacylated ghrelin.

According to another aspect, the present invention provides a method forscreening within a population of subjects suffering from diabetes,Prader-Willi Syndrome, obesity, insulin resistance or hyperphagia, whichsubjects are susceptible of benefiting from an administration ofunacylated ghrelin, a fragment thereof or an analog thereof, comprising:determining a level of circulating ghrelin and a ratio of circulatingghrelin/unacylated ghrelin from the subjects of the population;processing the level of circulating ghrelin and the ratio of circulatingghrelin/unacylated ghrelin at least in part based on a reference levelof circulating ghrelin and a reference ratio of circulatingghrelin/unacylated ghrelin to derive information conveying whether thelevel of circulating ghrelin and the ratio of circulatingghrelin/unacylated ghrelin are elevated; and causing conveyance of theinformation to a recipient for determining the subject's susceptibilityof benefiting from administration of unacylated ghrelin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of a study protocol involving Type2 Diabetes Mellitus (T2DM) subjects according to one embodiment of thepresent invention. SBM refers to Standard Breakfast Meal.

FIGS. 2A and 2B are graphs illustrating the effect of UAG infusion on AGserum levels in T2DM subjects. In FIG. 2A, AG serum levels in pg/ml weremeasured following placebo or UAG administration before and one hourafter SBM. In FIG. 2B, UAG serum levels in pg/ml were measured followingplacebo or UAG administration before and one hour after SBM.

FIGS. 3A and 3B are graphs illustrating the mean post-prandial glucoselevels in T2DM subjects as measured with a continuous glucose monitoringdevice (CGMS® iPro™ Continuous Glucose Recorder, Medtronic trading, TheNetherlands). FIG. 3A shows the mean absolute glucose levels after SBMand following placebo, UAG 3 mcg/kg/h and UAG 10 mcg/kg/h infusions.Repeated measures ANOVA p<0.0001; Bonferroni's Multiple Comparisonsplacebo vs. 3 mcg NS; placebo vs. 10 mcg p<0.001; 3 mcg vs. 10 mcgp<0.001. FIG. 3B shows the mean t0-t180 glucose levels for the threetreated groups.

FIGS. 4A and 4B are graphs illustrating the mean post-prandial glucoselevels in T2DM subjects. FIG. 4A shows the change in glucose levels frompre-meal baseline following placebo, UAG 3 mcg/kg/h and UAG 10 mcg/kg/hinfusions. Repeated measures ANOVA p<0.0001; Bonferroni's MultipleComparisons placebo vs. 3 mcg p<0.001; placebo vs. 10 mcg p<0.001; 3 mcgvs. 10 mcg p<0.05. FIG. 4B shows the mean t0-t180 glucose levels for thethree treated groups.

FIGS. 5A and 5B are graphs illustrating the peak plasma glucose levelsafter SBM in the eight subjects of the study following placebo, UAG 3mcg/kg/h and UAG 10 mcg/kg/h infusions. Wilcoxon matched-pairs signedrank test; *: 10 mcg vs. Placebo, p<0.05.

FIGS. 6A, 6B and 6C are graphs indicating the existence of a correlationbetween fasting basal AG and/or UAG concentrations and glycemic responseto UAG administration. The graph in FIG. 6A illustrates the correlationbetween fasting basal AG levels and change (following UAG vs. placeboadministration) in peak glucose levels after SBM. The graph in FIG. 6Billustrates the correlation between fasting basal AG levels and change(following UAG vs. placebo administration) in AUC glucose levels afterSBM using iPro continuous glucose measurements. The graph in FIG. 6Cillustrates the correlation between the ratio of fasting basal AG overUAG levels and change (following UAG vs. placebo administration) in AUCglucose levels after SBM using iPro continuous glucose measurements.

FIG. 7A is a schematic representation of a Hyperinsulinemic-EuglycemicClamp study protocol in T2DM subjects according to a further embodimentof the present invention.

FIG. 7B is a graph illustrating the effects of UAG on the M-index,reflective of insulin sensitivity, during the protocol as schematized inFIG. 7A. The change in M-index from baseline was measured followingplacebo and UAG infusions. *: p<0.05, One-tailed Mann Whitney test.

FIGS. 7C and 7D are graphs illustrating the effects of a short UAGinfusion period on basal AG levels in T2DM subjects. FIG. 7C showscirculating AG levels before UAG infusion and following a 2.5 hour UAGinfusion. FIG. 7D shows the AG change from baseline after a 2.5 hour UAGinfusion ([AG] level at 12:00—[AG] levels at 9:30). Two-tailed Wilcoxonmatched-pairs signed rank test; *: p<0.05; **: p<0.01.

FIG. 8 is a graph illustrating the effects of cyclic UAG fragment (6-13)on AG-induced food intake over the indicated study period. *p<0.001:Kruskal-Wallis One Way ANOVA. Differences between groups were evaluatedby the Dunn's test.

DETAILED DESCRIPTION

The present invention stems from, but is not limited to, the findings bythe Inventors that administration of UAG decreases levels of circulatingAG in subjects with T2DM. The present invention further stems from thefindings that the higher the level of basal AG, the more important arethe effects of UAG on reducing AG levels and on reducing the deleteriouseffects associated with AG levels such as, for example, obesity,hyperglycemia, insulin resistance, fat deposition, hyperphagia andobesity associated with insulin resistance. The Inventors have alsofound that the higher the ratio of circulating AG/UAG, the moreefficient is UAG in decreasing such ratio and in suppressing thedeleterious effects associated with AG/UAG ratio.

In view of this, circulating AG level and circulating AG/UAG ratio mayeach be used as biomarkers for identifying a subject's likelihood ofresponding to and/or benefiting from administration of UAG. Thesebiomarkers may thus be used for identifying within a population ofsubjects suffering from a condition such as, but not limited to,obesity, diabetes, insulin resistance, Prader-Willi, hyperphagia andhyperghrelinemia, which of the subjects are likely to respond to and/orbenefit from administration of UAG. The higher the circulating AG levelsand/or the higher the circulating AG/UAG ratio in a subject, the morethis subject is likely to respond to and/or benefit from theadministration of UAG.

To this date, studies have reported that UAG counteracts the peripheralactions of ghrelin on, for example, glucose and insulin metabolisms. Thepresent study provides the first evidence that administration of UAGalso suppresses circulating ghrelin levels and provides the firstevidence of the existence of a correlation between the level ofcirculating AG and the efficacy of UAG in improving metabolic parametersaffected by AG levels and/or by AG/UAG ratio.

The surprising demonstrations presented therein allow to expand theapplications and the indications for which unacylated ghrelin can beused so as to include the facilitation, amelioration and/or treatment ofconditions that result from AG levels and/or AG/UAG ratio.

These demonstrations also allow to expand the applications and theindications for which unacylated ghrelin can be used so as to includethe facilitation, amelioration and/or treatment of conditions thatresult from elevated AG levels and/or elevated AG/UAG ratio.

As used herein, the expression “elevated AG level(s)” refers to a levelof circulating AG that is above the AG level observed in normal and/orhealthy subjects. In some implementations, the expression “elevated AGlevel(s)” refers to a level of circulating AG at which one or moredeleterious physiological symptoms associated with AG appear, persist orare worsen in a subject.

As used herein, the expression “elevated AG/UAG ratio” refers to a ratioof circulating AG/UAG that is above the AG/UAG ratio observed in normaland/or healthy subjects. In some implementations, the expression“elevated AG/UAG ratio” refers to a ratio of circulating AG/UAG at whichone or more deleterious physiological symptoms associated with theAG/UAG ratio appear or persist or are worsen in a subject.

It is to be understood that several factors may affect the levels ofcirculating ghrelin and unacylated ghrelin in normal subjects. Examplesof such factors include, but are not limited to, gender, age, fitness,body mass index (BMI), eating habits, etc.

As used herein, the expression “inhibition of ghrelin” refers to animpairment of the biological activity of ghrelin which occurs due to adecrease in ghrelin levels and/or due to an impairment of its biologicalactivity.

A person skilled in the art will be familiar with the techniques andassays for measuring AG and UAG levels in a subject. Such techniques mayinclude techniques that involve the use of protease inhibitors such as4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) or othercocktail of protease inhibitors.

In one implementation of this embodiment, an obese subject ischaracterized as having a body weight approximately 20%, approximately25%, approximately 30% or greater than the normal body weight for saidsubject. Normal body weight may be determined by a comparison of theweight of the subject at a prior point in time, such as when AG levelswere normal and/or when AG/UAG ratio was normal, or by a comparison ofthe AG levels and/or AG/UAG ratio of the subject as compared to averagesof other subjects of a similar age and/or condition.

In another implementation of this embodiment, an overweight subject ischaracterized as having a body weight approximately 5% greater toapproximately 20% greater than the normal body weight for said subject.Normal body weight may be determined by a comparison of the weight ofthe subject at a prior point in time, such as as when AG levels werenormal and/or when AG/UAG ratio was normal, or by a comparison of the AGlevels and/or AG/UAG ratio as compared to averages of other subjects ofa similar age and/or condition.

In another implementation of this embodiment, a normal subject ischaracterized as having a body weight approximately 5% greater than toapproximately 5% less than the normal body weight for said subject.Normal body weight may be determined by a comparison of the weight ofthe subject at a prior point in time, such as when AG levels were normaland/or when AG/UAG ratio was normal, or by a comparison of the AG levelsand/or AG/UAG ratio as compared to averages of other subjects of asimilar age and/or condition. A normal weight subject may have a BMI inthe approximate range of 19-22.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the invention pertains.

-   -   i) Unacylated ghrelin, fragments and analogs thereof

The terms “unacylated ghrelin”, “des-acyl ghrelin” and the abbreviation“UAG” are intended to mean peptides that have the amino acid sequencespecified in SEQ ID NO: 1 which amino acid sequence is:

(SEQ ID NO: 1) Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys- Leu-Gln-Pro-Arg

Unacylated ghrelin may also be referred to as UAG (1-28).

Naturally-occurring variations of UAG include peptides that containsubstitutions, additions or deletions of one or more amino acids whichresult due to discrete changes in the nucleotide sequence of theencoding ghrelin gene or alleles thereof or due to alternative splicingof the transcribed RNA. It is understood that the changes do notsubstantially affect the properties, pharmacological and biologicalcharacteristics of unacylated ghrelin variants.

Those peptides may be in the form of salts. Particularly the acidicfunctions of the molecule may be replaced by a salt derivative thereofsuch as, but not limited to, a trifluoroacetate or an acetate salt.

By “peptide”, “polypeptide” or “protein” is meant any chain of aminoacids, regardless of length or post-translational modification (e.g.,glycosylation or phosphorylation), or chemical modification, or thosecontaining unnatural or unusual amino acids such as D-Tyr, ornithine,amino-adipic acid. The terms are used interchangeably in the presentapplication.

The expressions “fragments” and “fragments thereof” refer to amino acidfragments of a peptide such as UAG.

Fragments of UAG are shorter than the amino acid sequence depicted inSEQ ID NO: 1, therefore are shorter than 28 amino acid residues.Fragments of UAG may therefore be 27, 26, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 amino acidresidues in length. For example, fragments of UAG may have the aminoacid sequences depicted in Table 1 below:

TABLE 1 SEQ ID Fragment NO: Amino Acid Sequence UAG (1-14) 2Gly-Ser-Ser-Phe-Leu- Ser-Pro-Glu-His-Gln- Arg-Val-Gln-Gln UAG (1-18) 3Gly-Ser-Ser-Phe-Leu- Ser-Pro-Glu-His-Gln- Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser UAG (1-5) 4 Gly-Ser-Ser-Phe-Leu UAG (17-28) 5Glu-Ser-Lys-Lys-Pro- Pro-Ala-Lys-Leu-Gln- Pro-Arg UAG (6-13) 6Ser-Pro-Glu-His-Gln- Arg-Val-Gln UAG (8-13) 7 Glu-His-Gln-Arg-Val- GlnUAG (8-12) 8 Glu-His-Gln-Arg-Val UAG (6-18) 9 Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg- Lys-Glu-Ser UAG (8-11) 10 Glu-His-Gln-ArgUAG (9-12) 11 His-Gln-Arg-Val UAG (9-11) 29 His-Gln-Arg UAG (14-1) 30Gln Gln Val Arg Gln His Glu Pro Ser Leu Phe Ser Ser Gly

Any other fragments of UAG that preserve the biological activity of UAGare encompassed by the present invention. Some UAG fragments have beenreported in U.S. Pat. No. 8,222,217; U.S. Pat. No. 8,318,664 and inWO/2008/145749, incorporated herein in their entirety by reference,wherein it has been demonstrated that the smallest UAG fragment toretain the biological activity of UAG is UAG (9-12) depicted herein asSEQ ID NO: 11.

In one embodiment, the polypeptides such as UAG, fragments or analogsthereof, are used in a form that is “purified”, “isolated” or“substantially pure”. The polypeptides are “purified”, “isolated” or“substantially pure” when they are separated from the components thatnaturally accompany them. Typically, a compound is substantially purewhen it is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99%, by weight, of the total material in a sample.

The expressions “analog of unacylated ghrelin”, “analog of fragments ofunacylated ghrelin” and “analogs thereof” refer to both structural andfunctional analogs of UAG or fragments thereof which are, inter alia,capable of replacing UAG in the biological function of UAG as describedin the present application, such as, but not limited to modulate AG;inhibit AG; decrease circulating AG levels; decrease circulatingelevated AG levels; decrease circulating AG/UAG ratio; decreasecirculating elevated AG/UAG ratio; ameliorate the symptoms induced by AGlevels and/or AG/UAG levels; facilitate, prevent and/or treat conditionsassociated with circulating AG and/or circulating AG/UAG ratio andfacilitate, prevent and/or treat conditions associated with elevatedcirculating AG and/or elevated circulating AG/UAG ratio. Some analogs ofUAG have been reported in U.S. Pat. No. 8,222,217; U.S. Pat. No.8,318,664 and in WO/2008/145749, incorporated herein in their entiretyby reference.

Simple structural analogs comprise peptides showing homology with UAG asset forth in SEQ ID NO: 1 or homology with any fragment thereof. Anexample of an analog of AG is an isoform of Ghrelin-28, des Gln-14Ghrelin (a 27 amino acid peptide possessing serine 3 modification byn-octanoic acid) which is shown to be present in stomach. It isfunctionally identical to AG in that it binds to GHSR-1a with similarbinding affinity, elicits Ca²⁺ fluxes in cloned cells and induces GHsecretion with similar potency as Ghrelin-28. It is expected that UAGalso has a des Gln-14 UAG that is functionally identical to UAG.

Preferred analogs of UAG and preferred analogs of fragments of UAG arethose that vary from the native UAG sequence or from the native UAGfragment sequence by conservative amino acid substitutions; i.e., thosethat substitute a residue with another of like characteristics. Typicalsubstitutions include those among Ala, Val, Leu and Ile; among Ser andThr; among the acidic residues Asp and Glu; among Asn and Gln; among thebasic residues Lys and Arg; and among the aromatic residues Phe and Tyr.Particularly preferred are analogs in which several, for example, butnot limited to, 5-10, 1-5, or 1-2 amino acids are substituted, deleted,or added in any combination. For example, the analogs of UAG may differin sequence from UAG by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions (preferably conservative substitutions), deletions, oradditions, or combinations thereof.

There are provided herein, analogs of the peptides of the invention thathave at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence homology or sequence identity with the aminoacid sequences described herein over its full length, and sharing atleast one of the metabolic effects or biological activity of UAG. Aperson skilled in the art would readily identify an analog sequence ofunacylated ghrelin or an analog sequence of a fragment of unacylatedghrelin.

Examples of analogs of UAG are provided in Table 2 below:

TABLE 2 SEQ ID Analog NO: Amino acid sequence (Asp)8 UAG (6-13)NH₂ 12Ser-Pro-Asp-His- Gln-Arg-Val-Gln (Lys)11 UAG (6-13)NH₂ 13Ser-Pro-Glu-His- Gln-Lys-Val-Gln (Gly)6 UAG (6-13)NH₂ 14Gly-Pro-Glu-His- Gln-Arg-Val-Gln (Ala)6 UAG (6-13)NH₂ 15Ala-Pro-Glu-His- Gln-Arg-Val-Gln (Ala)7 UAG (6-13)NH₂ 16Ser-Ala-Glu-His- Gln-Arg-Val-Gln (Ala)8 UAG (6-13)NH₂ 17Ser-Pro-Ala-His- Gln-Arg-Val-Gln (Ala)9 UAG (6-13)NH₂ 18Ser-Pro-Glu-Ala- Gln-Arg-Val-Gln (Ala)10 UAG (6-13)NH₂ 19Ser-Pro-Glu-His- Ala-Arg-Val-Gln (Ala)11 UAG (6-13)NH₂ 20Ser-Pro-Glu-His- Gln-Ala-Val-Gln (Ala)12 UAG (6-13)NH₂ 21Ser-Pro-Glu-His- Gln-Arg-Ala-Gln (Ala)13 UAG (6-13)NH₂ 22Ser-Pro-Glu-His- Gln-Arg-Val-Ala (Acetyl-Ser)6 UAG 23 Ac-Ser-Pro-Glu-(6-13)NH₂ His-Gln-Arg-Val- Gln (Acetyl-Ser)6, (DPro)7 24 Ac-Ser-pro-Glu-UAG (6-13)NH₂ His-Gln-Arg-Val- Gln Cyclo (6-13) UAG (also 25Ser-Pro-Glu-His- referred to as cyclic Gln-Arg-Val-Gln UAG (6-13))(cycl) Cyclo (8,11), Lys 11, 26 Ser-Pro-Glu-His- UAG (6-13)amideGln-Lys-Val-Gln- amide Cyclo (8,11), Acetyl- 27 Ac-Ser-Pro-Glu-Ser6, Lys 11, UAG His-Gln-Lys-Val- (6-13)-amide Gln (cycl)Acetyl-Ser6, Lys 11, 28 Ac-Ser-Pro-Glu- UAG (6-13)NH₂ His-Gln-Lys-Val-Gln-NH₂

Analogs of UAG or analogs of fragments thereof are, for example, analogsobtained by alanine scans, by substitution with D-amino acids or withsynthetic amino acids or by cyclization of the peptide. Analogs of UAGor fragments thereof may comprise a non-naturally encoded amino acid,wherein the non-naturally encoding amino acid refers to an amino acidthat is not one of the common amino acids or pyrrolysine orselenocysteine, or an amino acid that occur by modification (e.g.post-translational modification) of naturally encoded amino acid(including, but not limited to, the 20 common amino acids or pyrrolysineand selenocysteine) but are not themselves incorporated into a growingpolypeptide chain by the translation complex. Examples of suchnon-naturally-occurring amino acids include, but are not limited to,N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine andO-phosphotyrosine.

As used herein, the term “modified” refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide.

The term “post-translational modification” refers to any modification ofa natural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as incell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

Examples of post-translational modifications are, but are not limitedto, glycosylation, acetylation, acylation, amidation, carboxylation,phosphorylation, PEGylation, addition of salts, amides or esters, inparticular C-terminal esters, and N-acyl derivatives of the peptides ofthe invention. The types of post-translational modifications are wellknown.

Certain peptides according to the present invention may also be incyclic form, such that the N- or C-termini are linked head-to-taileither directly, or through the insertion of a linker moiety, suchmoiety itself generally comprises one or more amino acid residues asrequired to join the backbone in such a manner as to avoid altering thethree-dimensional structure of the peptide with respect to thenon-cyclic form. Such peptide derivatives may have improved stabilityand bioavailability relative to the non-cyclized peptides.

Examples of cyclic peptides of the present invention include: cyclic UAG(1-14), cyclic UAG (1-18), cyclic UAG (17-28), cyclic UAG (6-13), cyclicUAG (8-13), cyclic UAG (8-12), cyclic UAG (8-11), cyclic UAG (9-12) andcyclic UAG (9-11) as well as the peptides identified in Table 2.

Methods for cyclizing peptides are well known in the art and for examplemay be accomplished by disulfide bond formation between two side chainfunctional groups, amide or ester bond formation between one side chainfunctional group and the backbone a-amino or carboxyl function, amide orester bond formation between two side chain functional groups, or amidebond formation between the backbone α-amino and carboxyl functions.These cyclization reactions have been traditionally carried out at highdilution in solution. Cyclization is commonly accomplished while thepeptide is attached to the resin. One of the most common ways ofsynthesizing cyclic peptides on a solid support is by attaching the sidechain of an amino acid to the resin. Using appropriate protectionstrategies, the C-and N-termini can be selectively deprotected andcyclized on the resin after chain assembly. This strategy is widelyused, and is compatible with either tert-butyloxycarbonyl (Boc) or9-fluorenylmethoxycarbonyl (Fmoc) protocols. However, it is restrictedto peptides that contain appropriate side chain functionality to attachto the solid support. A number of approaches may be used to achieveefficient synthesis of cyclic peptides. One procedure for synthesizingcyclic peptides is based on cyclization with simultaneous cleavage fromthe resin. After an appropriate peptide sequence is assembled by solidphase synthesis on the resin or a linear sequence is appended to resin,the deprotected amino group can react with its anchoring active linkageto produce protected cyclic peptides. In general, a final deprotectionstep is required to yield the target cyclic peptide.

Lactamazation, a form of cyclization, may be performed to form a lactambridge using Fmoc synthesis, amino acids with different protectinggroups at the lateral chains may be introduced, such as, but not limitedto, aspartic acid (or glutamic) protected with allyl ester at the betaester (or gamma ester for glutamic acid) and lysine protected withallyloxy carbamate at the N-ε. At the end of the synthesis, with theN-terminus of the peptide protected with Fmoc, Boc or other protectinggroup different from Alloc, the allyl and alloc protecting groups ofaspartic acid and lysine may be deprotected with, for example, palladium(0) followed by cyclization using PyAOP(7-Azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium-hexafluorophosphate) to produce the lactam bridge.

Unless otherwise indicated, an amino acid named herein refers to theL-form. Well recognized abbreviations in the art will be used todescribe amino acids, including levorotary amino acids (L-amino acids orL or L-form) and dextrorotatory amino acids (D-amino acids or D orD-form), Alanine (Ala or A), Arginine (Arg or R), Asparagine (Asn or N),Aspartic acid (Asp or D), Cysteine (Cys or C), Glutamic acid (Glu or E),Glutamine (Gln or Q), Glycine (Gly or G), Histidine (His or H),Isoleucine (Ile or I), Leucine (Leu or L), Lysine (Lys or K), Methionine(Met or M), Phenylalanine (Phe or F), Proline (Pro or P), Serine (Ser orS), Threonine (Thr or T), Tryptophan (Trp or W), Tyrosine (Tyr or Y) andValine (Val or V). An L-amino acid residue within the native peptidesequence may be altered to any one of the 20 L-amino acids commonlyfound in proteins or any one of the corresponding D-amino acids, rareamino acids, such as, but not limited to, 4-hydroxyproline orhydroxylysine, or a non-protein amino acid, such as P-alanine orhomoserine.

UAG peptides or fragments or analogs thereof may also be part of afusion protein. It is often advantageous to include an additional aminoacid sequence such as a signal sequence which contains for examplesecretory or leader sequences, pro-sequences, linker sequences which,inter alia, aid in purification such as multiple histidine residues(HA-tag), or an additional sequence for stability during recombinantproduction. The additional amino acids or sequence may be linked to atthe N-terminal or at the C-terminal of the polypeptide or may be linkedto any amino acid of the sequences located between the N- and theC-terminal to give rise the UAG peptides or fragment or analogs thereofhaving a linker moiety.

Any other analogs of UAG or fragments thereof or any other modified UAGor fragments thereof that preserve the biological activity of the fulllength UAG are encompassed by the present invention.

General methods and synthetic strategies used in providing functionaland structural analogs of UAG or fragments thereof are commonly used andwell known in the art and are described in publications such as:“Peptide synthesis protocols” ed, M. W. Pennigton & B. M. Dunn. Methodsin Molecular Biology. Vol 35. Humana Press, NJ.,1994; “Solid phasepeptide synthesis” by Stewart and Young, W. h Freeman & Co., SanFrancisco, 1969 and Erickson and Merrifield; and “The Proteins” Vol. 2,p. 255 et seq. (Ed. Neurath and Hill), Academic Press, New York, 1976.

As used herein, the term “homology” refers to sequence similaritybetween two peptides while retaining an equivalent biological activity.Homology can be determined by comparing each position in the alignedsequences. A degree of homology between amino acid sequences is afunction of the number of identical or matching amino acids at positionsshared by the sequences so that a “homologous sequence” refers to asequence sharing homology and an equivalent function or biologicalactivity. Assessment of percent homology is known by those of skill inthe art.

Methods to determine homology, identity and similarity of peptides arecodified in publicly available computer programs. Preferred computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, the GCG program package,BLASTP, BLASTN, and FASTA. The BLAST X program is publicly availablefrom NCBI and other sources. The well known Smith Waterman algorithm mayalso be used to determine identity.

Preferred parameters for polypeptide sequence comparison include thefollowing:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970);Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992);

Gap Penalty: 12; Gap Length Penalty: 4.

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison, Wis. Theaforementioned parameters are the default parameters for amino acidsequence comparisons (along with no penalty for end gaps).

The polypeptides of the invention may be prepared in any suitable manneras known in the art. Such polypeptides include isolated naturallyoccurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides, or polypeptides produced by acombination of these methods. Means and methods for preparing suchpolypeptides are well known in the art.

Certain aspects of the invention use UAG polynucleotides. These includeisolated polynucleotides which encode the UAG polypeptides, fragmentsand analogs defined in the application. As used herein, the term“polynucleotide” refers to a molecule comprised of a plurality ofdeoxyribonucleotides or nucleoside subunits. The linkage between thenucleoside subunits can be provided by phosphates, phosphonates,phosphoramidates, phosphorothioates, or the like, or by nonphosphategroups as are known in the art, such as peptoid-type linkages utilizedin peptide nucleic acids (PNAs). The linking groups can be chiral orachiral. The oligonucleotides or polynucleotides can range in lengthfrom 2 nucleoside subunits to hundreds or thousands of nucleosidesubunits. While oligonucleotides are preferably 5 to 100 subunits inlength, and more preferably, 5 to 60 subunits in length, the length ofpolynucleotides can be much greater (e.g., up to 100). Thepolynucleotide may be any of DNA and RNA. The DNA may be in any form ofgenomic DNA, a genomic DNA library, cDNA derived from a cell or tissue,and synthetic DNA. Moreover, the present invention may, in certainaspects, use vectors which include bacteriophage, plasmid, cosmid, orphagemid.

The expressions “biological activity” or “biological property”, or theterm “activity” in reference to the polypeptides of the presentinvention, are used interchangeably herein and refer to thepharmacological, biological or cellular abilities of the polypeptides ofthe invention and include, but are not limited to, the capacity ofreplacing UAG in the biological functions of UAG as described in thepresent application, such as, but not limited to, modulating AG;inhibiting AG; decreasing circulating AG levels; decreasing circulatingelevated AG levels; decreasing circulating AG/UAG ratio; decreasingcirculating elevated AG/UAG ratio; ameliorating the symptoms induced byAG levels and/or AG/UAG levels; facilitating, preventing and/or treatingconditions associated with circulating AG and/or circulating AG/UAGratio and facilitating, preventing and/or treating conditions associatedwith elevated circulating AG and/or elevated circulating AG/UAG ratio.

-   -   ii) Therapeutic methods, uses and compositions

According to one embodiment, the modulation of ghrelin levels and/ormodulation of AG/UAG ratio in a subject is desirable when suchmodulation is beneficial to the subject. According to anotherembodiment, the present invention provides a method for decreasing AGlevels and/or decreasing AG/UAG ratio in a subject. In someimplementations of this embodiment, the subject demonstrate elevated AGlevels and/or an elevated AG/UAG ratio and the symptoms associated withAG levels and/or associated with AG/UAG ratio are worsen or exacerbatedin this subject.

In some implementations of these embodiments, the AG levels refer to thecirculating AG levels and the AG/UAG ratio refers to the circulatingAG/UAG ratio. In some further implementations of this embodiment, thesubject shows one or more of the following symptoms associated with AGlevels and/or associated with AG/UAG ratio: hyperglycemia, insulinresistance, reduced fat utilization, adiposity, increase food intake,weight gain and suppression of insulin secretion. In furtherimplementations of this embodiment, the subject suffers from one or moreof the following conditions associated with the symptoms defined above:diabetes (e.g., type 2 diabetes), Prader-Willi Syndrome (PWS), obesity,obesity associated with insulin resistance, hyperphagia andhyperghrelinemia. The method comprises administering an effective amountof UAG, fragments, analogs, pharmaceutical salts thereof, and/or anycombinations thereof to the subject.

According to another embodiment, the present invention provides a methodfor ameliorating and/or diminishing the symptoms associated with AGlevels and/or associated with AG/UAG ratio in a subject. In someimplementations of this embodiment, the subject demonstrate elevated AGlevels and/or an elevated AG/UAG ratio and the symptoms associated withAG levels and/or associated with the AG/UAG ratio are worsen orexacerbated in this subject.

The present invention thus also provides a method for amelioratingand/or diminishing the symptoms associated with elevated AG levelsand/or associated with elevated AG/UAG ratio in such subject.

The symptoms associated with elevated AG levels include, but are notlimited to, hyperglycemia, insulin resistance, reduced fat utilization,adiposity, increased food intake, weight gain and suppression of insulinsecretion. The symptoms associated with elevated AG/UAG ratio include,but are not limited to, insulin resistance. In some implementations oftheses embodiment, the subject suffers from one or more of the followingconditions associated with the symptoms defined above: diabetes (e.g.type 2 diabetes), Prader-Willi Syndrome (PWS), obesity, obesityassociated with insulin resistance, hyperphagia and hyperghrelinemia.The method comprises administering an effective amount of UAG,fragments, analogs, pharmaceutical salts thereof, and/or anycombinations thereof to the subject. In some further implementations ofthis embodiment, the AG levels refer to the circulating AG levels andthe AG/UAG ratio refers to the circulating AG/UAG ratio. The methodcomprises administering an effective amount of UAG, fragments, analogs,pharmaceutical salts thereof and/or any combinations thereof to thesubject.

According to yet another embodiment, the present invention provides fora method of ameliorating and/or treating a condition, a disorder or adisease associated with AG levels and/or associated with AG/UAG ratio.In some implementations of this embodiment, the AG levels refer to thecirculating AG levels and the AG/UAG ratio refers to the circulating

AG/UAG ratio. Conditions, disorders or diseases associated with AGlevels include, but are not limited to, diabetes (e.g., type 2diabetes), Prader-Willi Syndrome (PWS), obesity, obesity associated withinsulin resistance, hyperphagia and hyperghrelinemia. The methodcomprises administering an effective amount of UAG, fragments, analogs,pharmaceutical salts thereof and/or any combination thereof to thesubject.

As used herein, the term “hyperghrelinemia” refers to a pathologicalcondition caused by elevated circulating AG levels and/or by an elevatedcirculating AG/UAG ratio. Subjects suffering from hyperghrelinemiaexhibit symptoms such as, but not limited to, hyperglycemia, insulinresistance, decreased insulin secretion, decreased fat utilization,adiposity, weight gain and/or a combination thereof.

In some implementations of the present invention, the more the AG levelsare elevated in these subjects, the more pronounced are the symptomsinduced by elevated AG levels and the more efficient is UAG, fragments,analogs and/or pharmaceutical salts thereof in ameliorating and/ordiminishing these symptoms.

In some other implementations of the present invention, the more theAG/UAG ratio is elevated in these subjects, the more pronounced are thesymptoms induced by an elevated AG/UAG ratio and the more efficient isUAG, fragments, analogs and/or pharmaceutical salts thereof inameliorating and/or diminishing these symptoms.

According to yet another embodiment, the present invention provides fora method for counteracting the peripheral actions of AG as well as todecrease AG levels and to ameliorate the symptoms induced by AG levels.

To the extent that new and yet uncovered conditions, diseases anddisorders can be ameliorated, prevented and/or treated with thereduction in AG and/or a reduction in AG/UAG ratio, the methods of thepresent invention can be utilized with respect to those conditions,disorders and diseases.

In one implementation of these embodiments, the method includes the stepof administering an effective amount of UAG or of a polypeptide definedherein which shares the same potential therapeutic indication as UAGitself to the subject in need of such administration.

Such polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 1, or comprises any fragment or any analog thereof such as forexample, those described in the above tables.

The actions of UAG have previously been shown to be conserved byfragments UAG (6-13) (SEQ ID NO: 6), UAG (8-13) (SEQ ID NO: 7), UAG(8-12) (SEQ ID NO: 8), UAG (8-11) (SEQ ID NO: 12), UAG (9-12) (SEQ IDNO: 11) and UAG (9-11) (SEQ ID NO: 29). U.S. Pat. Nos. 8,222,217 and8,318,664, incorporated herein in their entirety, have shown that thesefragments retain the activity of UAG full length on glucose, insulin andlipid metabolisms. A peptide with the inverse sequence of UAG (1-14)(SEQ ID NO: 3) and named UAG (14-1) (SEQ IDNO: 30) was used as anegative control in the experiments testing UAG fragments. UAG (8-11)(SEQ ID NO: 10) was shown to be the smallest UAG fragment to retain UAGactivities. The results provided herein further indicate that UAGfragments, such as for example, UAG (6-13) (SEQ ID NO: 6) and cyclic UAG(6-13) (SEQ ID NO: 25) retain UAG's ability to decrease AG levels anddecrease AG/UAG ratio.

In a further embodiment, UAG, fragments and/or analogs thereof are usedto reduce the elevated AG levels associated with Prader-Willi Syndrome(PWS). People who suffer from PWS suffer from slowed development, severeobesity and an insatiable appetite. Their hunger is so strong that itoften requires custodial enforcement of food availability to avert earlydeath as a result of hyperphagia. AG concentrations in these subjectsare higher than normal. This correlation between hyperphagia andincrease AG levels is consistent with the known orexigenic effect of AG.The data present herein demonstrate that administration of UAG candecrease the elevated AG levels in PWS subjects. The methods of theinvention can be used to help patients with Prader-Willi syndrome reducetheir ghrelin levels to more normal/healthier levels, curb theirappetite, and/or ameliorate other manifestations of this disorder. Thisdecrease in AG is expected to translate into a decrease in appetite andin a subsequent reduction in fat mass associated with PWS.

As used herein, the term “hyperphagia” refers to excessive hunger andabnormally large intake of solids by mouth. Hyperphagic conditions mayoccur in association with for example, central nervous system (CNS)disorders including gangliocytoma of the third ventricle, hypothalmicastrocytoma, Kleine-Levin Syndrome, Froehlich's Syndrome, Parkinson'sDisease, genetic disorders including Praeder-Willi Syndrome, majorpsychiatric disorders including anxiety, major depressive disorder,depressive phase of bipolar disorder, seasonal affective disorder, andschizophrenia, psychotropic medication, including delta-9tetrahydrocannabinol, antidepressants and neuroleptics and sleepdisorders including sleep apnea. Hyperphagia may occur in psychiatricdisorders such as depression, anxiety and schizophrenia. In someembodiments, administration of UAG, fragments and/or analogs thereof mayreduces the hyperphagia associated with these conditions.

As used herein, the term “treatment” refers to both therapeutictreatments as well as to prophylactic measures. Those in need oftreatment include those already with the disorder, disease or conditionas well as those in which the disease, disorder or condition is to beprevented. Those in need of treatment are also those in which thedisorder, disease or condition has occurred and left after-effects orscars. Treatment also refers to administering a therapeutic substanceeffective to improve or ameliorate, diminish symptoms associated with adisease, a disorder or a condition to lessen the severity of or cure thedisease, disorder or condition, or to prevent the disease, disorder orcondition from occurring or reoccurring.

Studies have demonstrated a persistent increase in plasma AG levels upto one year following a diet-induced weight loss in obese subjects(Refs. 28 and 29). The reduction of elevated AG levels in these subjectscould thus prevent obesity relapse while the subject is under diet.Therefore, in yet a further embodiment, the present invention provides amethod for improving the efficacy of diet-induced weight loss and/or forpreventing weight gain following diet-induced weight loss in a subjectin need of weight loss or in need of maintaining a weight loss byadministering an effective amount of UAG, fragments, analogs and/orpharmaceutical salts thereof to the subject.

In a further implementation of this embodiment, UAG, fragments and/oranalogs thereof are administered at the onset of the diet program andpreferably, UAG, fragments and/or analogs thereof are also administeredthroughout the diet program.

It is a further aspect of the present invention to provide for anypharmaceutical composition incorporating at least one of thepolypeptides as defined herein.

For therapeutic and/or pharmaceutical uses, the polypeptides as definedherein may be formulated for, but not limited to, intravenous,subcutaneous, transdermal, topical, oral, buccal, sublingual, nasal,inhalation, pulmonary, or parenteral administration according toconventional methods. Intravenous injection may be by bolus or infusionover a conventional period of time. The polypeptides as defined hereinmay also be administered directly to a target site within a subjecte.g., by biolistic delivery to an internal or external target site or bycatheter to a site in an artery.

In one embodiment, the polypeptides defined herein are administered as abolus. Accordingly, in one implementation of this embodiment, themedicament is administered as a bolus prior to meal, wherein the boluscomprises an effective amount of UAG, a fragment and/or an analogthereof of a salt thereof. The bolus may be administered one, twice,three times or more daily or may be administered according to otherdosage regimens.

Suitable dosage regiments are determined taking into account factorswell known in the art such as, but not limited to, type of subject beingdosed, the age, the weight, the sex and the medical condition of thesubject, the route of administration, the desired affect, etc.

Active ingredients, such as the polypeptides defined herein, may beadministered orally as a suspension and can be prepared according totechniques well known in the art of pharmaceutical formulation and maycontain, but not be limited to, microcrystalline cellulose for impartingbulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners/flavoringagents. As immediate release tablets, these compositions may contain,but are not limited to microcrystalline cellulose, dicalcium phosphate,starch, magnesium stearate and lactose and/or other excipients, binders,extenders, disintegrants, diluents and lubricants. The activeingredients may be administered by way of a controlled-release deliverysystem.

Administered by nasal aerosol or inhalation formulations may beprepared, for example, as solutions in saline, employing benzyl alcoholor other suitable preservatives, absorption promoters to enhancebioavailability, employing fluorocarbons, and/or employing othersolubilizing or dispersing agents.

The polypeptides of the invention may be administered in intravenous(both bolus and infusion), intraperitoneal, subcutaneous, topical withor without occlusion, or intramuscular form. When administered byinjection, the injectable solution or suspension may be formulated usingsuitable non-toxic, parenteral-acceptable diluents or solvents, wellknown in the art.

The polypeptides of the invention may also be formulated for topicaladministration. The term “topical” as used herein includes any route ofadministration that enables the compounds to line the skin or mucosaltissues.

The formulation suitable for topical application may be in the form of,for example, cream, lotion, solution, gel, ointment, paste, plaster,paint, bioadhesive, or the like, and/or may be prepared so as to containliposomes, micelles, microparticles and/or microspheres. The formulationmay be aqueous, i.e., contain water, or may be non-aqueous andoptionally used in combination with an occlusive overlayer so thatmoisture evaporating from the body surface is maintained within theformulation upon application to the body surface and thereafter.

Ointments, as is well known in the art of pharmaceutical formulation,are semisolid preparations that are typically based on petrolatum orother petroleum derivatives. Formulations may also be prepared withliposomes, micelles, microparticles and/or microspheres. Liposomes aremicroscopic vesicles having a lipid wall comprising a lipid bilayer, andcan be used as drug delivery systems. Micelles are known in the art tobe comprised of surfactant molecules arranged so that their polar headgroups form an outer spherical shell, while the hydrophobic, hydrocarbonchains are oriented towards the center of the sphere, forming a core.Microparticles are particulate carrier systems in the micron size range,normally prepared with polymers, which can be used as delivery systemsfor drugs or vaccines that are usually trapped within the particles.Microspheres, similarly, may be incorporated into the presentformulations and drug delivery systems. Like liposomes and micelles,microspheres essentially encapsulate a drug or drug-containingformulation. Microspheres are generally, although not necessarily,formed from synthetic or naturally occurring biocompatible polymers, butmay also be comprised of charged lipids such as phospholipids.

Preparations of formulations suitable for topical administration arewell known in the art and described in the pertinent texts andliterature.

In general, pharmaceutical compositions will comprise at least one ofthe polypeptides of the invention together with a pharmaceuticallyacceptable carrier which will be well known to those skilled in the art.The compositions may further comprise for example, one or more suitableexcipients, diluents, fillers, solubilizers, preservatives, carriers,salts, buffering agents and other materials well known in the artdepending upon the dosage form utilized.

Methods of composition are well known in the art.

In the present context, the term “pharmaceutically acceptable carrier”is intended to denote any material, which is inert in the sense that itsubstantially does not have any therapeutic and/or prophylactic effectper se and that are non-toxic. A pharmaceutically acceptable carrier maybe added to the polypeptides of the invention with the purpose of makingit possible to obtain a pharmaceutical composition, which has acceptabletechnical properties.

Examples of such carriers include ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts, or electrolytes such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, and PEG.

Carriers for topical or gel-based forms of polypeptides includepolysaccharides such as sodium carboxymethylcellulose ormethylcellulose, polyvinylpyrrolidone, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, PEG, and wood waxalcohols.

The polypeptides used for in vivo administration must be sterile. Thismay be accomplished by filtration through sterile filtration membranes,prior to or following lyophilization and reconstitution. Thepolypeptides ordinarily will be stored in lyophilized form or insolution.

Therapeutic polypeptide compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

For use in the methods defined herein, the invention also provides anarticle of manufacture or a commercial package or kit, comprising: acontainer, a label on the container, and a composition comprising thepolypeptides of the invention as an active agent within the containerwhen used at the indicated level, wherein the composition is effectivefor, inter alia, modulating AG; inhibiting AG; decreasing circulating AGlevels; decreasing circulating elevated AG levels; decreasingcirculating AG/UAG ratio; decreasing circulating elevated AG/UAG ratio;ameliorating the symptoms induced by AG levels and/or AG/UAG levels;facilitating, preventing and/or treating conditions associated withcirculating AG and/or circulating AG/UAG ratio and/or facilitating,preventing and/or treating conditions associated with elevatedcirculating AG and/or elevated circulating AG/UAG ratio.

An “effective amount” or a “therapeutically effective amount” refers toan amount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result. A therapeutically effectiveamount of the peptides noted herein may vary according to factors suchas the disease state, age, sex, and weight of the individual, and theability of the compound to elicit a desired response in the individual.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the compound are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result, such as tomodulate AG; inhibit AG; decrease circulating AG levels; decreasecirculating elevated AG levels; decrease circulating AG/UAG ratio;decrease circulating elevated AG/UAG ratio; ameliorate the symptomsinduced by AG levels and/or AG/UAG levels; facilitate, prevent and/ortreat conditions associated with circulating AG and/or circulatingAG/UAG ratio and/or facilitate, prevent and/or treat conditionsassociated with elevated circulating AG and/or elevated circulatingAG/UAG ratio. A prophylactically effective amount can be determined asdescribed above for the therapeutically effective amount. For anyparticular subject, specific dosage regimens may be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thecompositions.

For example, a therapeutically effective amount or effective dose of thepeptides of the invention (also referred to herein as “active compound”)is an amount sufficient to modulate AG; inhibit AG; decrease circulatingAG levels; decrease circulating elevated AG levels; decrease circulatingAG/UAG ratio; decrease circulating elevated AG/UAG ratio; ameliorate thesymptoms induced by AG levels and/or AG/UAG levels; facilitate, preventand/or treat conditions associated with circulating AG and/orcirculating AG/UAG ratio and/or facilitate, prevent and/or treatconditions associated with elevated circulating AG and/or elevatedcirculating AG/UAG ratio. The methods and/or assays for measuring suchparameters are known to those of ordinary skill in the art.

The therapeutically effective amount of the invention will generallyvary from about 0.001 μg/kg to about 10 mg/kg, more particularly fromabout 0.01 μg/kg to about 10 mg/kg, and even more particularly fromabout 1 μg/kg to about 1 mg/kg. Therapeutically effective amounts oreffective doses that are outside this range but that have the desiredtherapeutic effect are also encompassed by the present invention.

In a one embodiment, the subject noted above is a mammal, in a furtheraspect, a human.

In a further embodiment, the present polypeptides may be administered incombination with additional pharmacologically active substances or maybe administered in combination with another therapeutic method. Thecombination may be in the form of a kit-in-part system, wherein thecombined active substances may be used for simultaneous, sequential orseparate administration.

-   -   iii) AG levels and AG/UAG ratio as biomarkers

According to another embodiment, the present invention relates to theuse of circulating AG levels and the use of circulating AG/UAG ratio asbiomarkers for the identification of subjects that are likely to respondto and/or benefit from a treatment comprising the administration of atherapeutically effective amount of UAG, a fragment thereof and/or ananalog thereof.

In one implementation of this embodiment, circulating AG levels and/orcirculating AG/UAG ratio are measured in a blood sample obtained from asubject according to known methods in the art. The level of circulatingAG and/or the ratio of circulating AG/UAG are then processed in partbased on reference circulating levels of AG and reference circulatingAG/UAG ratios (derived, for example, from the subject himself or from apopulation of control subjects) so as to derive information whichconveys whether the subject has an abnormal level of circulating AG(i.e., below or above a normal or healthy level) and/or an abnormalratio of circulating AG/UAG (i.e., below or above a normal or healthyratio). An information which conveys that the subject has an elevatedcirculating AG level and/or an elevated circulating AG/UAG ratioindicates that the subject is likely to respond to administration ofUAG, a fragment thereof or an analog thereof. Administration of UAG, afragment thereof or an analog thereof in such subject is thus likely todecrease circulating AG levels and/or circulating AG/UAG ratio as wellas to lessen the effects associated with elevated circulating AG levelsand elevated circulating AG/UAG ratio.

In another implementation of this embodiment, the subject suffers from acondition such as, but not limited to, diabetes (type 1 or type 2),obesity, Prader-Willy, insulin resistance, hyperphagia andhyperghrelinemia. The biomarkers defined herein may be used to determineif such subject is likely to response and/or is likely to benefit fromadministration of UAG, a fragment thereof or an analog thereof. Asubject suffering from such a condition and having an elevatedcirculating AG level and/or an elevated circulating AG/UAG ratio islikely to respond and/or benefit from administration of UAG, a fragmentthereof and/or an analog thereof.

In another implementation of this embodiment, the biomarkers may be usedfor identifying subjects within a population of subjects that are likelyto respond to and/or benefit from administration of a therapeuticallyeffective amount of UAG, a fragment thereof or an analog thereof. Inthis implementation, the subjects of the population suffer from acondition such as, but not limited to, diabetes (type I or type 2),Prader-Willi Syndrome (PWS), obesity, insulin resistance, hyperphagiaand hyperghrelinemia.

In accordance with another implementation of this embodiment, thepresent invention provides a method of displaying information conveyinga subject's likelihood of responding to and/or benefiting fromadministration of UAG, a fragment thereof and/or an analog thereof.

Such implementation may, for example, by carried out by an apparatus,such as, but not limited to, a computer readable storage medium storinga program element suitable for execution by a computer unit. In suchimplementation, the program element implements a graphical userinterface module which displays information conveying the subject'slikelihood of responding to and/or benefiting from administration ofUAG, a fragment thereof and/or an analog thereof.

The graphical user interface module is adapted for displaying a set ofuser modifiable information fields allowing a user to enter a set ofinformation data elements associated to the subject's level ofcirculating AG, level of circulating UAG and/or circulating AG/UAG ratiomeasured according to methods known in the art. The graphical userinterface module is also adapted for displaying a control allowing auser to cause the set of information data elements to be transmitted toa processing unit. The processing unit is adapted to derive a subject'slikelihood of responding to and/or benefiting from administration ofUAG, a fragment thereof and/or an analog thereof at least in part on thebasis of the set of information data elements. The graphical userinterface module receives the subject's level of circulating AG, levelof circulating UAG and/or circulating AG/UAG ratio and is adapted todisplay the subject's likelihood of responding to and/or benefiting fromadministration of UAG, a fragment thereof and/or an analog thereof withreference to normal or healthy circulating AG levels and/or normal orhealthy circulating AG/UAG ratio (derived, for example, from the subjector from a population of control subjects). Optionally, the graphicaluser interface module is adapted to display the subject's likelihood ofresponding to and/or benefiting from administration of UAG, a fragmentthereof and/or an analog thereof with reference to circulating AG levelsand/or circulating AG/UAG ratio observed in subjects suffering from acondition such as, but not limited to, diabetes (type I or type 2),Prader-Willi Syndrome (PWS), obesity, insulin resistance, hyperphagiaand hyperghrelinemia In accordance with a specific implementation, theset of information data elements may also comprise a gender component, aweight component, a body mass index (BMI) component, a fitnesscomponent. The set of information data elements may further include anyother suitable item of information associated with the subject.

In accordance with a specific implementation, the subject's likelihoodof responding to and/or benefiting from administration of UAG, afragment thereof and/or an analog thereof includes an indicative dataelement conveying a likelihood of responding to and/or benefiting fromadministration of UAG, a fragment thereof and/or an analog thereof. Theindicative data element may be expressed in the form of a score,likelihood, a percentile value or in any other format suitable.

In accordance with a specific implementation, the graphical userinterface module is adapted for displaying a graph conveying thelikelihood of a subject to respond to and/or to benefit fromadministration of AUG, a fragment thereof and/or an analog thereof, thegraph conveying: a first information indicative of a referencenormal/healthy level of circulating AG and/or a reference normal/healthycirculating AG/UAG ratio (optionally, the graphical user interfacemodule is adapted for displaying information indicative of referencecirculating AG levels and circulating AG/UAG ratios in subjectssuffering from a condition such as, but not limited to, diabetes (type Ior type 2), Prader-Willi Syndrome (PWS), obesity, insulin resistance,hyperphagia and hyperghrelinemia); a second information indicative ofthe subject's circulating level of AG, circulating level of UAG and/orcirculating AG/UAG ratio; and a third information conveying a likelihoodof the subject to respond to and/or to benefit from UAG administration

Experiments and Data Analysis

The data present herein reports a strong suppressing effect of UAG onserum AG levels and on serum AG/UAG ratio in T2DM subjects.

A continuous overnight (15 hours) infusion of two doses of UAG (3 μgUAG/kg/hr and 10 μg UAG/kg/hr) versus placebo in a cross-over model onthe AG concentrations was performed in eight overweight subjects withtype 2 diabetes. FIG. 1 depicts a schematic representation of the studyprotocol. Glucose and insulin responses to a standard breakfast meal(SBM) in the subjects and reasonable metabolic control were assessed.During the infusions with UAG, subjects did not report more side effectsthan placebo. Laboratory evaluations showed no significant changes inchemistry or parameters and the side effects were not dose-dependent.

Infusion of UAG Decreases Plasma AG Levels

The data present in FIGS. 2A and 2B show the changes in serum AG levels(FIG. 2A) and UAG levels (FIG. 2B) before and after SBM. Beforeinitiation of SBM, AG levels are significantly decreased from 21.01±8.9pg/ml (mean±SD) during placebo infusion to 3.0±6.7 pg/ml in the presence3 mcg/kg·hr UAG infusion and to 1.4±3.2 pg/ml in the presence of 10mcg/kg·hr UAG infusion. AG levels are also decreased following SMB from14.03±9.4 pg/ml in placebo to 0.8±1.8 pg/ml in the presence 3 mcg/kg·hrUAG infusion and to 0.8±1.8 pg/ml in the presence of 10 mcg/kg·hr UAGinfusion (FIG. 2A). In parallel, an overnight infusion of UAG resultedin an increase in UAG levels (FIG. 2B). UAG levels increased from105.9±31.4 (mean±SD) pg/ml in placebo infusion and before start of theSBM to 10998±2623 pg/ml in the presence 3 mcg/kg·hr UAG infusion and to12085±1616 pg/ml in the presence of 10 mcg/kg·hr UAG infusion. Theseresults indicate that administration of UAG reduces serum AG levels inT2DM subjects.

Infusion of UAG Fragment Counteracts AG-Induced Food Intake

A cyclized fragment of UAG, namely cyclic UAG (6-13) as depicted in SEQID NO: 25, was able to counteract the orexigenic effects induced by AGin rat (FIG. 8). These results demonstrate that UAG fragments as definedherein which retain the core sequence responsible for UAG-relatedactions/activities also retain their effects on AG levels. As discussedabove, there is a benefit of inhibiting the effect of AG or its levels(e.g. devcreasing food consumption) in certain subjects. These resultsdemonstrate a beneficial role for UAG in the treatment of patients withPrader Willi syndrome, in whom elevated AG levels are associated withhyperphagia. Inhibition of AG levels and/or biological effects shouldthus result in decreased appetite and/or food consumption.

Infusion of UAG Decreases Post-Prandial Plasma Glucose Levels

An overnight infusion of UAG significantly depressed post-prandialglucose levels as assessed by iPro continuous glucose monitoring (FIGS.3A and 3B). The area under the curves decreased from 1618 mmol/3 hrs forplacebo infusion to 1601 mmol/3 hrs and 1540 mmol/3 hrs for the 3 and 10mcg UAG infusions respectively. FIGS. 4A and 4B depict the resultsobtained as changes from the glucose pre-SBM baseline. The area underthe curves decreased for the 3 and 10 mcg UAG infusions doses whencompared to placebo. FIGS. 5A and 5B indicate a decrease in plasmaglucose peak after SBM when UAG is administered. An overall decrease inpost-prandial plasma glucose peaks can be observed in the subjects (FIG.5B). Overall, these data indicate that administration of UAG decreasespost-prandial plasma glucose levels in subjects with T2DM.

UAG-Induced Reduction in Plasma AG Levels and AG/UAG Ratio Correlateswith Reduction in Glucose Levels

The inventors were able to show the existence of a correlation betweenfasting AG levels and/or UAG levels and glycemic response following UAGadministration using a standard glucokinase assay. Further todemonstrating such correlation, FIG. 6A shows that the more elevated thefasting AG levels are, the best are the hypoglycemic effect of UAGadministration. FIG. 6B also shows the same correlation and effect usingiPro continuous glucose measurements. Fasting AG/UAG ratio alsocorrelates with the glycemic response following UAG administration (FIG.6C). The higher AG/AUG ratios correlate with the best hypoglycemiceffect of UAG infusion using iPro continuous glucose measurements.

Infusion of UAG Improves Insulin Sensitivity

Insulin sensitivity in T2DM subjects was assessed using thehyperinsulinemic-euglycemic clamp protocol as depicted in FIG. 7A. Inpatients receiving effective euglycemic insulin clamp at the start of a2.5 hr placebo/UAG infusion, the M-index change from baseline wasincreased by 36% in the UAG vs. placebo group (p=0.02) (FIG. 7B). Theseresults demonstrate that UAG infusion improves insulin resistance inT2DM subjects.

Shorter UAG Infusion is Sufficient to Decrease Plasma AG Levels

AG levels were measured in T2DM subjects prior to UAG infusion andfollowing a 2.5 hour-long UAG infusion. The data presented in FIG. 7Cshows that the shortened UAG infusion period was sufficient to decreaseplasma AG levels. FIG. 7D indicates the changes in AG levels frombaseline demonstrating that a short UAG infusion period suffices todecrease plasma AG levels.

These data demonstrate, inter alia, that administration of UAG improvesglucose levels during a SBM through a reduction in AG. A significantdecrease in peak glucose levels after meal was also observed. Nosignificant change in serum insulin levels during the infusion of lowand high dose of UAG infusions was observed (data not shown), whichindicates that the improved glycemic control correlates with an improvedinsulin sensitivity. The data also show that administration of UAGimproves hyperglycemia in a ghrelin concentration dependent manner thusmaking the UAG hypoglycemic effects stronger at higher AG levels or athigher AG/UAG ratio.

These results are the first indication that UAG is a potent inhibitor ofghrelin levels making UAG a strong candidate for the development of aghrelin inhibitor in the treatment of metabolic disorders.

Materials and Technical Protocols

Study design—Single-center, investigator initiated, double blind andplacebo controlled randomized study. During the first visit medicalhistory, medication use and vital signs of subjects were checked. Bloodsamples were also taken for chemistry and hematology analysis. The studyconsisted of three rounds of hospitalization (visit 2, 3 and 4) of twodays each.

The first day started at approximately 15:00 and continued until 14:00the next day. The eight subjects were divided into 3 groups for thestudy. The dosages used during these rounds were either 3 mcg/kg·hr UAG,10 mcg/kg·hr UAG or placebo/saline solution prepared by the hospitalpharmacy and delivered in 3 bags for each subject. Neither subjects norresearchers were aware of the drug given in these rounds. A washoutperiod of one week was performed between the treatment periods.

Before, during and after the study, blood samples were taken for, AG,UAG, chemistry, hematology and CAC cells. Blood samples for glucose werealso taken via i.v catheter. For continuous glucose monitoring aContinuous Glucose monitor (iPro2, Medtronic trading, The Netherlands)was placed in the abdomen of the subjects. Weight and blood pressurewere measured during each visit. A washout period of at least 1 week wasperformed between the treatment periods.

Subjects—Eight subjects were enrolled (2 females and 6 males; mean ageof 53 yrs (ranging from 31-65 years old) with mean body mass index (BMI)of 31.5 kg/m², range 26-36 kg/m². Seven of the eight subjects usedmetformin daily. All subjects were diagnosed with type 2 diabetes for atleast 3 months prior to enrollment. Metformin monotherapy for at least 3months prior to screening was allowed, but metformin treatment wasstopped 1 day prior to start of each treatment period. In the populationmean glycosylated hemoglobin level (HbA1c) was 52 mmol/mol range from 48mmol/mol to 57 mmol/mol (6.9%; range 6.5-7.4%).) and Body Mass Index wasabove 25 kg/m². Exclusion criteria consisted in history or presence oflong-term type 2 diabetes complications; clinically significantabnormalities in laboratory evaluation at screening, and use of systemiccorticosteroids within 60 days prior to screening. Prior to infusion,subjects received two indwelling catheters: the first catheter wasinserted prior to treatment; and the second catheter was inserted priorto the SBM for blood sampling. The second catheter was kept patent byslow infusion of isotonic saline.

Study drug—UAG used in this study was produced by Bachem AG,Hauptstrasse 144, Bubendorf CH-4416, Switzerland. UAG was delivered aslyophilized powder (vials containing 5 mg of drug) and stored at thelocal pharmacy according to the manufacturer's specifications.

Study procedures—Infusions were performed at the local clinical researchunit for 15 hours from 9 pm to 12 am. Each volume of UAG solution wasfiltered and diluted in 0.9% of saline solution to obtain theappropriate dose for administration. Placebo consisted in 0.9% of salinesolution. The dose was calculated based on the subject's weight. Eachvial was reconstituted with 5 ml water for injection, filtered through a0.22 μm filter. The dose was then injected in a 500 ml bag of 0.9% NaCl.Three bags were prepared to ensure a continuous 15-hour infusion at 100ml/hour.

Standard breakfast meal (SBM) consisted of:

-   -   3 slices wheat bread; p1 3 portions of margarine;    -   2 slices of cheese (48% fat);    -   1 portion of jam;    -   1 cup of whole milk; and    -   1 boiled egg;        for a total of 714 kcal (17% proteins; 46% fat; and 37%        carbohydrates). The SBM had to be consumed within 15 minutes,        from 8:00 am to 8:15 am. At each of the 3 visits, the following        safety parameters were assessed: hemoglobin, hematocrit,        platelet count, WBC count, RBC count, and differential and were        determined using Sysmex XE 2100, Firma Sysmex, Ecustraat 11,        4879 NP Etten-Leur. AST, ALT, alkaline phosphatase, total        bilirubin, creatine phophokinase (CPK), lactate dehydrogenase        (LDH), creatinine, urea, amylase, lipase, uric acid, glucose,        cholesterol, LDL, HDL, triglycerides, sodium, potassium,        calcium, chloride, protein and albumin are determined using the        Hitachi Modular P800, Roche Transistorstraat 41, 1332 CK Almere.        Blood glucose levels were measured using a continuous glucose        monitoring device (Medtronic CGMS iPro™ Continuous Glucose        Recorder, Medtronic; The Netherlands) that was subcutaneously        inserted for the whole treatment period. Serum glucose levels        (using the in-house glucokinase assay) were also assessed every        30 minutes and starting before, and continuing for 4 hours        after, the SBM. During the CGMS, all subjects had to perform at        least four capillary glycemic tests per day. The data collected        were entered into the CGMS monitor to obtain correlation        coefficients between the SMBG and the CGMS values. All SMBG        tests were performed using a digital glucometer (Contour,        Bayer). AG and UAG levels were assessed before the start of the        overnight infusion, 10 minutes before the start and 30 minutes        after the SBM. To preserve acylation of ghrelin, blood samples        were collected directly into EDTA tubes, then within 2 minutes 1        ml of EDTA-blood was added to 1 ml of preservative solution on        ice (0.0295 N HCl containing 72 mM NaCl, 58 mM NaF, 4 mM        4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (a water        soluble, irreversible serine protease inhibitor; AEBSF), pH 3.0,        295 mOsm/KgPlasma was prepared by centrifugation at 4° C., then        1 ml was acidified with 100 μl 1N HCl and stored at −80° C.        until assays were performed.

The ghrelin assay utilized MSD ELISA 96-well plates (Meso ScaleDiscovery (MSD), Gaithersburg, MD, USA) coated by incubation with 30μl/well of capture antibody (D4 diluted to 1 μg/ml in PBS;anti-C-terminal ghrelin (Gutierrez et al. 2008 PNAS 105:6320-6325)),overnight at room temperature. The capture antibody was removed andwells were blocked with 150 μl casein buffer (Pierce) for 1 h at roomtemperature with shaking. Standards for

AG and UAG were prepared using eight 4× serial dilutions in caseinbuffer starting at 8 ng/ml and 30 ng/ml, respectively. Preserved plasmawas diluted 1:1 in casein buffer. Separate plates were used fordetection of AG and UAG. Standards and samples (25 μl/well) were loadedonto coated ELISA plates, and incubated at room temperature with shakingfor 2 hours, washed 3× with PBS-T (150 μl/well). The C2-4a1 and E8detection antibodies (N-terminal AG and N-terminal UAG, respectively)were sulfotagged using the standard protocol from MSD. They were thendiluted 1:10000 in 0.2× casein/0.05% Tween 20 and added to AG or UAGplates, respectively, at 25 μl/well. Plates were incubated at roomtemperature for 1 hour with shaking. Plates were washed three times withPBS-T (150 μl/well). Finally, 150 μl of 1× Read Buffer (MSD) was addedto each well, and the plates were immediately read on an MSD SectorImager 6000. AG and UAG values for samples were calculated byinterpolation from their respective standard curves using Sector Imagersoftware.

Clamp study—Randomized 2-period, 2-treatment, double-blind study of UAGvs. vehicle infusion, evaluating one dose (10 μg/kg/h) of UAGadministrated by continuous iv infusion for 2.5 hrs. In patientsreceiving effective euglycemic insulin clamp at the start of a 2.5 hrplacebo/UAG infusion.

Statistical analyses—Data analyses were performed with the GraphPadPrism 5.0 (GraphPad Software, Inc. La Jolla, Calif. 92037 USA). Theresults are given as means (±SE). Comparisons were calculated usingBonferroni's Multiple Comparisons, Wilcoxon matched-pairs signed-ranktests and ANOVA analyses.

With respect to the experimental data presented in FIG. 8,Sprague-Dawley rats of 7 weeks of age, weighing between 275 g and 300 g,were fed a pellet diet ad libitum and were singly housed in plasticcage. The experiment was performed at 2.5 h after the onset of the lightcycle in freely fed rats. The rates were i.p. injected simultaneouslywith vehicle plus vehicle, vehicle+AG (13 μg/kg) or cyclised UAG (6-13)(SEQ ID NO: 25) (42 μg/kg)+AG (13 μg/kg). Immediately after thecompletion of the i.p. injection in rats, the night-ad-libitum food wasremoved and replaced by 2 pellets for each animal, previously weighed,placed into the top of the cage. Food intake was calculated as thedifference between the food weight before and after the feeding periodat each time interval (30 min, 1 h, and 2 h). Cumulative food intake wascalculated by summating the values of the different time periods.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

All documents mentioned in the specification are herein incorporated byreference.

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1. A method for decreasing ghrelin levels in a subject, comprising administering an effective amount of unacylated ghrelin, a fragment thereof, an analog thereof and/or pharmaceutically acceptable salts thereof to the subject.
 2. The method as defined in claim 1, wherein the ghrelin levels are elevated circulating ghrelin levels.
 3. (canceled)
 4. The method as defined in claim 1, wherein the ghrelin levels are associated with at least one of hyperglycemia, insulin resistance, reduced fat utilization, adiposity, weight gain or suppression of insulin secretion in the subject.
 5. The method as defined in claim 1, wherein the subject suffers from a condition selected from the group consisting of type 2 diabetes, hyperghrelinemia, obesity associated with insulin resistance and Prader-Willi Syndrome and any combination thereof.
 6. The method as defined in claim 1, for decreasing a circulating ghrelin/unacylated ghrelin ratio in the subject.
 7. The method as defined in claim 6, wherein the circulating ghrelin/unacylated ghrelin ratio is an elevated circulating ghrelin/unacylated ghrelin ratio. 8-25. (canceled)
 26. A method for preventing weight gain in a subject following diet-induced weight loss, comprising administering an effective amount of unacylated ghrelin, a fragment thereof, an analog thereof and/or pharmaceutically acceptable salts thereof to the subject.
 27. The method as defined in claim 26, wherein the administration of the unacylated ghrelin, the fragment thereof, the analog thereof and/or the pharmaceutically acceptable salts thereof decreases the ghrelin levels.
 28. (canceled)
 29. The method as defined in claim 1, wherein the unacylated ghrelin has an amino acid sequence as set forth in SEQ ID NO:
 1. 30. The method as defined in claim 1, wherein the unacylated ghrelin fragment comprises an amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO:
 25. 31. The method as defined in claim 1, wherein the unacylated ghrelin fragment is as set forth in SEQ ID NO:
 6. 32. The method as defined in claim 1, wherein the unacylated ghrelin fragment is as set forth in SEQ ID NO: 6 and comprises a linker moiety.
 33. The method as defined in claim 1, wherein the unacylated ghrelin fragment is as set forth in SEQ ID NO:
 25. 34. The method as defined in claim 1, wherein the effective amount is from about 0.001 μg/kg to about 10 μg/kg.
 35. The method as defined in claim 1, wherein effective amount is from about 1 μg/kg to about 1 mg/kg. 36-37. (canceled)
 38. A pharmaceutical composition comprising an effective amount of unacylated ghrelin, a fragment thereof, an analog thereof and/or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable diluent for decreasing ghrelin levels in a subject. 39-48. (canceled)
 49. The pharmaceutical composition as defined in claim 38, wherein the unacylated ghrelin fragment is as set forth in SEQ ID NO:
 25. 50-58. (canceled)
 59. A method for screening within a population of subjects suffering from diabetes, Prader-Willi Syndrome, obesity, insulin resistance or hyperphagia, which subjects within the population are susceptible of benefiting from an administration of unacylated ghrelin, a fragment thereof or an analog thereof, comprising: determining a level of circulating ghrelin from the subjects of the population; processing the level of circulating ghrelin at least in part based on a reference level of circulating ghrelin to derive information conveying whether the level of circulating ghrelin is elevated; and causing conveyance of the information to a recipient for determining the subject's susceptibility of benefiting from administration of unacylated ghrelin, a fragment thereof or an analog thereof.
 60. The method of claim 59, further comprising: determining a ratio of circulating ghrelin/unacylated ghrelin from the subjects of the population; processing the ratio of circulating ghrelin/unacylated ghrelin at least in part based on a reference level of circulating ghrelin/unacylated ghrelin to derive information conveying whether the ratio of circulating ghrelin/unacylated ghrelin is elevated; and causing conveyance of the information to a recipient for determining the subject's susceptibility of benefiting from administration of unacylated ghrelin.
 61. The method as defined claim claim 59, further comprising administering a therapeutically effective amount of unacylated ghrelin, a fragment thereof, an analog thereof and/or pharmaceutically acceptable salts thereof to the subjects that are susceptible of benefiting from administration of unacylated ghrelin.
 62. (canceled)
 63. The method as defined in claim 60, wherein the ratio of circulating ghrelin/unacylated ghrelin is elevated when it is above the normal ratio of circulating ghrelin/unacylated ghrelin.
 64. The method as defined in claim 59, wherein the subjects of the population suffer from Type 2 diabetes. 